Detection of varicella-zoster virus

ABSTRACT

The invention provides methods to detect VZV in biological samples using real-time PCR. Primers and probes for the detection of VZV are provided by the invention. Articles of manufacture containing such primers and probes for detecting VZV are further provided by the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority under 35 U.S.C.§120 to U.S. application Ser. No. 09/945,203, filed Aug. 31, 2001, nowU.S. Pat. No. 6,849,407 which claims the benefit under 35 U.S.C. §119(e)of U.S. application Ser. No. 60/229,499, filed Aug. 31, 2000.

TECHNICAL FIELD

This invention relates to viral diagnostics, and more particularly todetection of varicella- zoster virus (VZV).

BACKGROUND

The most common dermal manifestation resulting from primary infectionwith varicella-zoster virus (VZV) is chickenpox (varicella), whichgenerally occurs in early childhood. Reactivation of latent virus occursin about 10-20% of adults and produces vesicles that are typicallyconfined to a single dermatome of the skin (herpes zoster). VZVinfections can cause systemic infections of the central nervous andrespiratory systems in immunologically competent patients and producedisseminated disease of multiple organ systems in those with impairedimmunologic defenses. Laboratory diagnosis is important fordistinguishing herpes simplex virus (HSV) from VZV infections sinceclinical presentation of herpes zoster due to VZV can be confused withthe dermal distribution produced by HSV.

SUMMARY

The invention provides for methods of identifying varicella-zoster virus(VZV) in a biological sample. Primers and probes for detecting VZV areprovided by the invention, as are kits containing such primers andprobes. Methods of the invention can be used to rapidly identify VZV DNAfrom specimens for diagnosis of VZV infection and to differentiate VZVinfections from HSV infections. Using specific primers and probes, themethods include amplifying and monitoring the development of specificamplification products using fluorescence resonance energy transfer(FRET).

In one aspect of the invention, there is provided a method for detectingthe presence or absence of VZV in a biological sample from anindividual. The method to detect VZV includes performing at least onecycling step, which includes an amplifying step and a hybridizing step.The amplifying step includes amplifying a portion of a VZV gene 28nucleic acid molecule from the biological sample using a pair of gene 28primers, thereby producing a gene 28 amplification product. Thehybridizing step includes annealing a pair of gene 28 probes to the gene28 amplification product. Generally, the members of the pair of gene 28probes hybridize within no more than five nucleotides of each other. Afirst gene 28 probe of the pair of gene 28 probes is typically labeledwith a donor fluorescent moiety and a second gene 28 probe of the pairof gene 28 probes is labeled with a corresponding acceptor fluorescentmoiety. The method further includes detecting the presence or absence ofFRET between the donor fluorescent moiety of the first gene 28 probe andthe acceptor fluorescent moiety of the second gene 28 probe uponhybridization of the pair of gene 28 probes to the amplificationproduct. The presence of FRET is usually indicative of the presence ofVZV in the biological sample, while the absence of FRET is usuallyindicative of the absence of VZV in the biological sample.

Alternatively, the amplifying step includes amplifying a portion of aVZV gene 29 nucleic acid molecule from the biological sample using apair of gene 29 primers, thereby producing a gene 29 amplificationproduct. The hybridizing step includes annealing a pair of gene 29probes to the gene 29 amplification product. Generally, the members ofthe pair of gene 29 probes hybridize within no more than fivenucleotides of each other. A first gene 29 probe of the pair of gene 29probes is typically labeled with a donor fluorescent moiety and a secondgene 29 probe of the pair of gene 29 probes is labeled with acorresponding acceptor fluorescent moiety. The method further includesdetecting the presence or absence of FRET between the donor fluorescentmoiety of the first gene 29 probe and the acceptor fluorescent moiety ofthe second gene 29 probe upon hybridization of the pair of gene 29probes to the gene 29 amplification product. The presence of FRET isusually indicative of the presence of VZV in the biological sample,while the absence of FRET is usually indicative of the absence of VZV inthe biological sample. The methods to detect VZV using gene 28 and gene29 can be performed individually, sequentially or concurrently.

A pair of gene 28 primers generally includes a first gene 28 primer anda second gene 28 primer. The first gene 28 primer can include thesequence 5′-GAC AAT ATC ATA TAC ATG GAA TGT G-3′ (SEQ ID NO:1), and thesecond gene 28 primer can include the sequence 5′-GCG GTA GTA ACA GAGAAT TTC TT-3′ (SEQ ID NO:2). A first gene 28 probe can include thesequence 5′-CGA AAA TCC AGA ATC GGA ACT TCT T-3′ (SEQ ID NO:3), and thesecond gene 28 probe can include the sequence 5′-CCA TTA CAG TAA ACT TTAGGC GGT C-3′ (SEQ ID NO:4).

A pair of gene 29 primers generally includes a first gene 29 primer anda second gene 29 primer. The first gene 29 primer can include thesequence 5′-TGT CCT AGA GGA GGT TTT ATC TG-3′ (SEQ ID NO:5), and thesecond gene 29 primer can include the sequence 5′-CAT CGT CTG TAA GACTTA ACC AG-3′ (SEQ ID NO:6). A first gene 29 probe can include thesequence 5′-GGG AAA TCG AGA AAC CAC CCT ATC CGA C-3′ (SEQ ID NO:7), andthe second gene 29 probe can include the sequence 5′-AAG TTC GCG GTA TAATTG TCA GTG GCG-3′ (SEQ ID NO:8). In some aspects, one of the gene 28 orgene 29 primers can be labeled with a fluorescent moiety (either a donoror acceptor, as appropriate) and can take the place of the gene 28 orgene 29 probes, respectively.

The members of the pair of gene 28 probes can hybridize within no morethan two nucleotides of each other, or can hybridize within no more thanone nucleotide of each other. A representative donor fluorescent moietyis fluorescein, and corresponding acceptor fluorescent moietes includeLC-Red 640, LC-Red 705, Cy5, and Cy5.5. Additional corresponding donorand acceptor fluorescent moieties are known in the art.

In one aspect, the detecting step includes exciting the biologicalsample at a wavelength absorbed by the donor fluorescent moiety andvisualizing and/or measuring the wavelength emitted by the acceptorfluorescent moiety (i.e., visualizing and/or measuring FRET). In anotheraspect, the detecting step includes quantitating the FRET. In yetanother aspect, the detecting step can be performed after each cyclingstep (e.g., in real-time).

Generally, the presence of FRET within 50 cycles (e.g., 20, 25, 30, 35,40, or 45 cycles) indicates the presence of a VZV infection in theindividual. In addition, determining the melting temperature between oneor both of the gene 28 probe(s) and the gene 28 amplification orsimilarly one or both of the gene 29 probe(s) and the gene 29amplification product can confirm the presence or absence of the VZV.

Representative biological sample include dermal swabs, cerebrospinalfluid, ganglionic tissue, brain tissue, ocular fluid, blood, sputum,bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine.The above-described methods can further include preventing amplificationof a contaminant nucleic acid. Preventing amplification can includeperforming the amplifying step in the presence of uracil and treatingthe biological sample with uracil-DNA glycosylase prior to amplifying.

In addition, the cycling step can be performed on a control sample. Acontrol sample can include the same portion of the VZV gene 28 nucleicacid molecule. Alternatively, a control sample can include a nucleicacid molecule other than a VZV gene 28 nucleic acid molecule. Cyclingsteps can be performed on such a control sample using a pair of controlprimers and a pair of control probes. The control primers and probes areother than gene 28 primers and probes. One or more amplifying stepsproduces a control amplification product. Each of the control probeshybridizes to the control amplification product.

In another aspect of the invention, there are provided articles ofmanufacture, or kits. Kits of the invention can include a pair of gene28 primers, and a pair of gene 28 probes, and a donor and correspondingacceptor fluorescent moieties. For example, the first gene 28 primerprovided in a kit of the invention can have the sequence 5′-GAC AAT ATCATA TAC ATG GAA TGT G-3′ (SEQ ID NO:1) and the second gene 28 primer canhave the sequence 5′-GCG GTA GTA ACA GAG AAT TTC TT-3′ (SEQ ID NO:2).The first gene 28 probe provided in a kit of the invention can have thesequence 5′-CGA AAA TCC AGA ATC GGA ACT TCT T-3′ (SEQ-ID NO:3) and thesecond gene 28 probe can have the sequence 5′-CCA TTA CAG TAA ACT TTAGGC GGT C-3′ (SEQ ID NO:4). Articles of manufacture of the invention canfurther or alternatively include a pair of gene 29 primers, a pair ofgene 29 probes, and a donor and corresponding acceptor fluorescentmoieties. For example, the first gene 29 primer provided in a kit of theinvention can have the sequence 5′-TGT CCT AGA GGA GGT TTT ATC TG-3′(SEQ ID NO:5), and the second gene 29 primer can have the sequence5′-CAT CGT CTG TAA GAC TTA ACC AG-3′ (SEQ ID NO:6). The first gene 29probe provided in a kit of the invention can have the sequence 5′-GGGAAA TCG AGA AAC CAC CCT ATC CGA C-3′ (SEQ ID NO:7), and the second gene29 probe can have the sequence 5′-AAG TTC GCG GTA TAA TTG TCA GTG GCG-3′(SEQ ID NO:8). Articles of manufacture can include fluorophoric moietiesfor labeling the probes or probes already labeled with donor andcorresponding acceptor fluorescent moieties. The article of manufacturecan also include a package insert having instructions thereon for usingthe primers, probes, and fluorophoric moieties to detect the presence orabsence of VZV in a biological sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

DETAILED DESCRIPTION

A real-time assay for detecting VZV in a biological sample that is moresensitive than existing assays is described herein. Primers and probesfor detecting VZV infections and articles of manufacture containing suchprimers and probes are provided by the invention. The increasedsensitivity of real-time PCR for detection of VZV compared to othermethods, as well as the improved features of real-time PCR includingsample containment and real-time detection of the amplified product,make feasible the implementation of this technology for routinediagnosis of VZV infections in the clinical laboratory.

Varicella-zoster Virus (VZV)

Varicella-zoster virus (VZV) is a human alphaherpesvirus (HHV-3) with arespiratory port of entry. VZV causes two distinct diseases, varicellaand herpes zoster. Varicella infection causes the contagious childhooddisease chicken pox. The initial infection results in virus growth inthe regional lymph nodes that then feeds a primary viremia within 24hours of contact. Subsequent sites of viral replication are establishedin organs such as the spleen and liver. A secondary lymphocyte-mediatedviremia targets the subcutaneous epithelial cells where furtherreplication causes deep necrotic lesions of the epidermis and dermis. Astrong humoral and cellular immunity clears all actively replicating andantigen-presenting VZV infected cells from the bloodstream, skin andganglia. Immunity is protective against subsequent exposure VZV. Inaddition, a varicella vaccine has been developed and is used tovaccinate children (e.g., between the ages of 12 months and 12 years)against chickenpox.

The varicella virus also can penetrate the peripheral nervous system andremain latent in dorsal root ganglia for many years. VZV activation inadults produces herpes zoster, commonly known as shingles. Herpes zosterinfections present as a deep vascularized skin rash that is typicallyrestricted to a dermatome. The latent state molecular biology of VZV,including viral load in ganglionic tissue is being investigated using,for example, PCR.

Each VZV virion contains one molecule of linear double-stranded DNA. VZVcontains one of the smallest genomes in the herpesvirus family, with 125kb of potential coding sequence. The VZV genome contains at least 69functional genes based upon the identification of open reading frames(ORFs). The VZV genome is typical of the alphaherpesviruses with theORFs arranged in the same order, position, and relative direction as theORFs from, for example, pseudorabiesvirus (PRV), herpes simplex virus-1(HSV-1) and equine herpesvirus (EHV-1).

VZV Nucleic Acids and Oligonucleotides

The invention provides methods to detect VZV by amplifying, for example,a portion of the VZV gene 28 or gene 29 nucleic acid. VZV nucleic acidsother than those exemplified herein (e.g., other than gene 28 and gene29) also can be used to detect VZV in a sample and are known to those ofskill in the art. The nucleic acid sequence of the VZV genome, as wellas VZV gene 28 (encoding DNA polymerase) and gene 29 (encoding asingle-stranded binding protein), are available (see, for example,GenBank Accession Nos. X04370, M14891 and M16612). Note that gene 28 hasbeen referred to as UL30 in the literature while gene 29 has beenreferred to as UL29. Specifically, primers and probes to amplify anddetect VZV gene 28 nucleic acid molecules are provided by the inventionas are primers and probes to amplify and detect VZV gene 29 nucleic acidmolecules.

Primers that amplify a VZV nucleic acid molecule, e.g., VZV gene 28 orgene 29, can be designed using, for example, a computer program such asOLIGO (Molecular Biology Insights, Inc., Cascade, Colo.). Importantfeatures when designing oligonucleotides to be used as amplificationprimers include, but are not limited to, an appropriate sizeamplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 15 to30 nucleotides in length.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that FRET can occur (e.g., within no more than 1, 2, 3, or 4nucleotides of each other). This minimal degree of separation typicallybrings the respective fluorescent moieties into sufficient proximitysuch that FRET occurs. It is to be understood, however, that otherseparation distances (e.g., 6 or more nucleotides) are possible providedthe fluorescent moieties are appropriately positioned relative to eachother (for example, with a linker arm) such that FRET can occur. Inaddition, probes can be designed to hybridize to targets that contain apolymorphism or mutation, thereby allowing differential detection of VZVstrains based on either absolute hybridization of different pairs ofprobes corresponding to the particular VZV strain to be distinguished ordifferential melting temperatures between, for example, members of apair of probes and each amplification product corresponding to a VZVstrain to be distinguished. As with oligonucleotide primers,oligonucleotide probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are generally 15 to 30 nucleotides inlength.

Constructs of the invention include vectors containing a VZV nucleicacid molecule, e.g., VZV gene 28 or gene 29, or fragment thereof and canbe used, for example, as a control template nucleic acid molecule.Vectors suitable for use in the present invention are commerciallyavailable and/or produced by recombinant DNA technology methods routinein the art. VZV gene 28 or gene 29 nucleic acid molecules can beobtained, for example, by chemical synthesis, direct cloning from VZV,or by PCR amplification. A VZV nucleic acid molecule or fragmentsthereof can be operably linked to a promoter or other regulatory elementsuch as an enhancer sequence, a response element, or an inducibleelement that modulates expression of the VZV nucleic acid molecule. Asused herein, operably linking refers to connecting a promoter and/orother regulatory elements to a VZV nucleic acid molecule in such a wayas to permit and/or regulate expression of the VZV nucleic acidmolecule. A promoter that does not normally direct expression of VZVgene 28 or gene 29 can be used to direct transcription of a gene 28 orgene 29 nucleic acid using, for example, a viral polymerase, a bacterialpolymerase, or a eukaryotic RNA polymerase II. Alternatively, the gene28 or gene 29 native promoter can be used to direct transcription of agene 28 or gene 29 nucleic acid, respectively, using, for example, a VZVRNA polymerase enzyme. In addition, operably linked can refer to anappropriate connection between a VZV gene 28 or gene 29 promoter orregulatory element and a heterologous coding sequence (i.e., a non-gene28 or gene 29 coding sequence, for example, a reporter gene) in such away as to permit expression of the heterologous coding sequence.

Constructs suitable for use in the methods of the invention typicallyinclude, in addition to VZV gene 28 or gene 29 nucleic acid molecules,sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, add anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

Constructs of the invention containing VZV gene 28 or gene 29 nucleicacid molecules can be propagated in a host cell. As used herein, theterm host cell is meant to include prokaryotes and eukaryotes such asyeast, plant and animal cells. Prokaryotic hosts may include E. coli,Salmonella typhimurium, Serratia marcescens and Bacillus subtilis.Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichiapastoris, mammalian cells such as COS cells or Chinese hamster ovary(CHO) cells, insect cells, and plant cells such as Arabidopsis thalianaand Nicotiana tabacum. A construct of the invention can be introducedinto a host cell using any of the techniques commonly known to those ofordinary skill in the art. For example, calcium phosphate precipitation,electroporation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer are common methods for introducingnucleic acids into host cells. In addition, naked DNA can be delivereddirectly to cells (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in the present invention include oligonucleotidescapable of acting as a point of initiation of nucleic acid synthesiswithin VZV gene 28 or gene 29. A primer can be purified from arestriction digest by conventional methods, or it can be producedsynthetically. The primer is preferably single-stranded for maximumefficiency in amplification, but the primer can be double-stranded.Double-stranded primers are first denatured, i.e., treated to separatethe strands. One method of denaturing double stranded nucleic acids isby heating.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

If the VZV template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min.

If the double-stranded nucleic acid is denatured by heat, the reactionmixture is allowed to cool to a temperature that promotes annealing ofeach primer to its target sequence on the VZV nucleic acid. Thetemperature for annealing is usually from about 35° C. to about 65° C.The reaction mixture is then adjusted to a temperature at which theactivity of the polymerase is promoted or optimized, i.e., a temperaturesufficient for extension to occur from the annealed primer to generateproducts complementary to the template nucleic acid. The temperatureshould be sufficient to synthesize an extension product from each primerthat is annealed to a nucleic acid template, but should not be so highas to denature an extension product from its complementary template(e.g., the temperature generally ranges from about 40° to 80° C.).

PCR assays can employ VZV nucleic acid such as DNA or RNA, includingmessenger RNA (mRNA). The template nucleic acid need not be purified; itmay be a minor fraction of a complex mixture, such as VZV nucleic acidcontained in human cells. DNA or RNA may be extracted from a biologicalsample such as dermal swabs, cerebrospinal fluid, ganglionic tissue,brain tissue, ocular fluid, blood, sputum, bronchio-alveolar lavage,bronchial aspirates, lung tissue, and urine by routine techniques suchas those described in Diagnostic Moleculear Microbiology: Principles andApplications (Persing et al. (eds), 1993, American Society forMicrobiology, Washington D.C). Nucleic acids can be obtained from anynumber of sources, such as plasmids, or natural sources includingbacteria, yeast, viruses, organelles, or higher organisms such as plantsor animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target VZV nucleic acid molecule. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donor and acorresponding acceptor fluorescent moiety are positioned within acertain distance of each other, energy transfer takes place between thetwo fluorescent moieties that can be visualized or otherwise detectedand/or quantitated. As used herein, two oligonucleotide probes, eachcontaining a fluorescent moiety, can hybridize to an amplificationproduct at particular positions determined by the complementarily of theoligonucleotide probes to the VZV target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productnucleic acid at the appropriate positions, a FRET signal is generated.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Förster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 μm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthamide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm for the purpose of the present invention is the distance inAngstroms (Π) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 to about 25 Π. The linker arm maybe of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

An acceptor fluorescent moiety such as an LC™-Red 640-NHS-ester can becombined with C6-Phosphoramidites (available from ABI (Foster City,Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC™-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of VZV

Detection of VZV can be by a number of testing modalities and from anumber of specimen types. Standard laboratory diagnosis has beenobtained by culture of the virus in diploid fibroblasts seeded intoshell vial cell cultures or by immunostaining viral antigens in infectedcells collected by swabs of vesicles from patients. In addition,serologic assays for immunoglobulin G or A class antibodies also areused to detect and diagnose VZV, although serological assays may be lessuseful for diagnosing VZV infections in view of available varicellavaccinations. See, for example, Brinker & Doern, 1993, Diagn. Microbiol.Infect. Dis., 17:75-77; Coffin & Hodinka, 1995, J. Clin. Microbiol.,33:2792-2795; McCarter & Ratkiewicz, 1998, Am. J. Clin. Pathol.,109:631-633; Shirm et al., 1989, J. Med. Virol., 28:1-6; and Sauerbreiet al., 1999, J. Clin. Virol., 14:31-6 for detection of VZV.

Conventional PCR methods also have been used to detect VZV. ConventionalPCR-based amplification is generally followed by transfer to a solidsupport and detection using a labeled probe (e.g., a Southern orNorthern blot). These methods are labor intensive and frequently requiremore than one day to complete. Additionally, the manipulation ofamplification products for the purposes of detection (e.g., by blotting)increases the risk of carry-over contamination and false positives. Byusing commercially available real-time PCR instrumentation (e.g.,LightCycler™, Roche Molecular Biochemicals, Indianapolis, Ind.), PCRamplification and detection of the amplification product can be combinedin a single closed cuvette with dramatically reduced cycling time. Sincedetection occurs concurrently with amplification, the real-time PCRmethods obviate the need for manipulation of the amplification product,and diminish the risk of cross-contamination between amplificationproducts. Real-time PCR greatly reduces turn-around time and is anattractive alternative to conventional PCR techniques in the clinicallaboratory.

The present invention provides methods for detecting the presence orabsence of VZV in a biological sample from an individual. Methodsprovided by the invention avoid problems of sample contamination, falsenegatives, and false positives. The methods include performing at leastone cycling step that includes amplifying a VZV portion of a gene 28nucleic acid molecule from a biological sample using a pair of gene 28primers. Each of the gene 28 primers anneals to a target within oradjacent to a VZV gene 28 nucleic acid molecule such that at least aportion of the amplification product contains nucleic acid sequencecorresponding to gene 28 and, more importantly, such that theamplification product contains the nucleic acid sequences that arecomplementary to the gene 28 probes. The gene 28 amplification productis produced provided that VZV nucleic acid is present. Each cycling stepfurther includes hybridizing a pair of gene 28 probes to the gene 28amplification product. According to the invention, one of the gene 28probes is labeled with a donor fluorescent moiety and the other islabeled with a corresponding acceptor fluorescent moiety. The presenceor absence of FRET between the donor fluorescent moiety of the firstgene 28 probe and the corresponding acceptor fluorescent moiety of thesecond gene 28 probe is detected upon hybridization of both gene 28probes to the gene 28 amplification product.

Each cycling step includes an amplification step and a hybridizationstep, and each cycling step is usually followed by a FRET detectingstep. Multiple cycling steps are performed, preferably in athermocycler. The above-described methods for detecting VZV in abiological sample using primers and probes directed toward gene 28 alsocan be performed using other VZV gene-specific primers and probes, forexample, gene 29-specific primers and gene 29-specific probes.

As used herein, “amplifying” refers to the process of synthesizingnucleic acid molecules that are complementary to one or both strands ofa template nucleic acid molecule (e.g., VZV gene 28 or gene 29 nucleicacid molecules). Amplifying a nucleic acid molecule typically includesdenaturing the template nucleic acid, annealing primers to the templatenucleic acid at a temperature that is below the melting temperatures ofthe primers, and enzymatically elongating from the primers to generatean amplification product. Amplification typically requires the presenceof deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,Platinum® Taq) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

If amplification of VZV nucleic acid occurs and an amplification productis produced, the step of hybridizing results in a detectable signalbased upon FRET between the members of the pair of probes. As usedherein, “hybridizing” refers to the annealing of probes to anamplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

Generally, the presence of FRET indicates the presence of VZV in thebiological sample, and the absence of FRET indicates the absence of VZVin the biological sample. Inadequate specimen collection, transportationdelays, inappropriate transportation conditions, or use of certaincollection swabs (calcium alginate or aluminum shaft) are all conditionsthat can affect the success and/or accuracy of a test result, however.

Using the methods disclosed herein, detection of FRET within 30 cyclingsteps is indicative of a VZV infection. Samples in which FRET isdetected after more than 30 cycling steps also is indicative of a VZVinfection, but can be evaluated for VZV infection, if desired, using amethod of the invention with a different gene target or an assay otherthan the real-time PCR described herein. The cycle number at which FRETis detectable can be correlated with the amount of VZV in a biologicalsample and, hence, in the individual (e.g., viral load).

Methods of the invention also can be used for VZV vaccine efficacystudies or epidemiology studies. For example, an attenuated VZV in avaricella vaccine can be detected using the methods of the inventionduring the time when virus is still present in an individual. For suchvaccine efficacy studies, the methods of the invention can be used todetermine, for example, the replicating ability or persistence of anattenuated virus used in a vaccine, or can be performed in conjunctionwith an additional assay such as a serologic assay to monitor anindividual's immune response to such a vaccine. In addition, methods ofthe invention can be used to distinguish one VZV strain from another forepidemiology studies of, for example, the origin or severity of anoutbreak of varicella (chickenpox) and/or herpes zoster (shingles).

Representative biological samples that can be used in practicing themethods of the invention include dermal swabs, cerebrospinal fluid,ganglionic tissue, brain tissue, ocular fluid, blood, sputum,bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine.Biological sample collection and storage methods are known to those ofskill in the art. Biological samples can be processed (e.g., by nucleicacid extraction methods and/or kits known in the art) to release VZVnucleic acid or in some cases, the biological sample is contacteddirectly with the PCR reaction components and the appropriateoligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Ssimilarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the gene 28 or gene 29 probes from therespective amplification product can confirm the presence or absence ofVZV in the sample.

Within each thermocycler run, control samples are cycled as well.Positive control samples can amplify VZV nucleic acid control template(other than gene 28 or gene 29) using, for example, control primers andcontrol probes. Positive control samples can also amplify, for example,a plasmid construct containing VZV gene 28 or gene 29 nucleic acidmolecule. Such a plasmid control can be amplified internally (e.g.,within the biological sample) or in a separate sample run side-by-sidewith the patients' samples. Each thermocycler run should also include anegative control that, for example, lacks VZV template DNA. Suchcontrols are indicators of the success or failure of the amplification,hybridization and/or FRET reaction. Therefore, control reactions canreadily determine, for example, the ability of primers to anneal withsequence-specificity and to initiate elongation, as well as the abilityof probes to hybridize with sequence-specificity and for FRET to occur.

In an embodiment, the methods of the invention include steps to avoidcontamination. For example, an enzymatic method utilizing uracil-DNAglycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and5,945,313 to reduce or eliminate contamination between one thermocyclerrun and the next. In addition, standard laboratory containment practicesand procedures are desirable when performing methods of the invention.Containment practices and procedures include, but are not limited to,separate work areas for different steps of a method, containment hoods,barrier filter pipette tips and dedicated air displacement pipettes.Consistent containment practices and procedures by personnel arenecessary for accuracy in a diagnostic laboratory handling clinicalsamples.

Although conventional PCR methods in conjunction with FRET technologycan be used to practice the methods of the invention, in one embodiment,a LightCycler™ instrument is used. A detailed description of theLightCycler™ System and real-time and on-line monitoring of PCR can befound at http://biochem.roche.com/lightcycler. The following patentapplications describe real-time PCR as used in the LightCycler™technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™instrument is a rapid thermal cycler combined with a microvolumefluorometer utilizing high quality optics. This rapid thermocyclingtechnique uses thin glass cuvettes as reaction vessels. Heating andcooling of the reaction chamber are controlled by alternating heated andambient air. Due to the low mass of air and the high ratio of surfacearea to volume of the cuvettes, very rapid temperature exchange ratescan be achieved within the LightCycler™ thermal chamber. Addition ofselected fluorescent dyes to the reaction components allows the PCR tobe monitored in real time and on-line. Furthermore, the cuvettes serveas an optical element for signal collection (similar to glass fiberoptics), concentrating the signal at the tip of the cuvette. The effectis efficient illumination and fluorescent monitoring of microvolumesamples.

The LightCycler™ carousel that houses the cuvettes can be removed fromthe instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorometer, as part of the LightCycler™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvette. Fluorescent light emitted from thesample is then focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LightCycler™instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468)includes three band-pass filters (530 nm, 640 nm, and 710 nm), providingthree-color detection and several fluorescence acquisition options. Datacollection options include once per cycling step monitoring, fullycontinuous single-sample acquisition for melting curve analysis,continuous sampling (in which sampling frequency is dependent on samplenumber) and/or stepwise measurement of all samples after definedtemperature interval.

The LightCycler™ can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 msec. After each cycling step, a quantitative display offluorescence vs. cycle number can be continually updated for allsamples. The data generated can be stored for further analysis.

The fluorescent formats currently used as reporters in the LightCycler™System are the double-stranded DNA binding dye SYBR Green I, andfluorescent labels attached to hybridization probes. Whereas signaldevelopment in the presence of the SYBR Green I dye is dependent on theformation of double-stranded DNA (regardless of the DNA sequence),signal from hybridization requires the production of sequence-specificprobes. Hybridization probes are typically labeled with two differentfluorescent moieties and hybridize in close proximity to each other in atarget DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler™ Instrument. The fluorescein canthen transfer its energy to an acceptor fluorescent moiety, for example,LightCycler™-Red 640 (LC™-Red 640) or LightCycler™-Red 705 (LC™-Red705), during FRET. The acceptor fluorescent moiety then emits light of alonger wavelength, which is then detected by the optical detectionsystem of the LightCycler™ instrument. Efficient FRET can only takeplace when the fluorescent moieties are in direct local proximity andwhen the emission spectrum of the donor fluorescent moiety overlaps withthe absorption spectrum of the acceptor fluorescent moiety. Theintensity of the emitted signal can be correlated with the number oforiginal target DNA molecules (e.g., the number of VZV genomes).

In another embodiment, and ABI PRISM® 7700 Sequence Detection System(Applied Biosystems, Foster City, Calif.) also is suitable forperforming the methods described herein for detecting VZV. Informationon PCR amplification and detection using an ABI PRISM® 770 system can befound at http://www.appliedbiosystems.com/products. The presentinvention, however, is not limited by the configuration of acommercially available instrument.

Articles of Manufacture

The invention further provides for articles of manufacture to detectVZV. An article of manufacture according to the present invention caninclude primers and probes used to detect VZV, together with suitablepackaging materials. Representative primers and probes for detection ofVZV are complementary to VZV gene 28 or gene 29 nucleic acid molecules.Methods of designing primers and probes are disclosed herein, andrepresentative examples of primers and probes that amplify and hybridizeto VZV gene 28 or gene 29 nucleic acid molecules are provided.

Articles of manufacture of the invention also can include one or morefluorscent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor fluorescent moiety for labeling one ofthe gene 28 or gene 29 probes and an acceptor fluorescent moiety forlabeling the other gene 28 or gene 29 probe. Examples of suitable FRETdonor fluorescent moieties and corresponding acceptor fluorescentmoieties are provided above.

Articles of manufacture of the invention also can contain a packageinsert having instructions thereon for using the gene 28 primers andprobes or gene 29 primers and probes to detect VZV in a biologicalsample. Articles of manufacture may additionally include reagents forcarrying out the methods disclosed herein (e.g., buffers, polymeraseenzymes, co-factors, or agents to prevent contamination). Such reagentsmay be specific for one of the commercially available instrumentsdescribed herein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Specimens, Cell Cultures and Nucleic Acid Extractions

Dermal swabs (n=253) from patients suspected of having VZV infectionswere extracted and inoculated into MRC-5 shell vial cell cultures aspreviously described for HSV (Gleaves et al., 1985, J. Clin. Microbiol.,21:29-32). Nucleic acids were extracted (IsoQuick, Orca Research, Inc.,Bothell, Wash.) and amplified by LightCycler™ PCR (Espy et al., 2000, J.Clin. Microbiol., 38:795-799).

Example 2 LightCycler™ PCR

The LightCycler™ instrument can amplify target nucleic acids withinabout 30-40 min and monitors the development of PCR product byfluorescence assay after each cycling step (amplification andhybridization). All samples were amplified by LightCycler™ PCR withprimers directed to both gene 28 and gene 29. PCR primers for detectionof VZV DNA using gene 28 were designed using the OLIGO program and hadthe following sequences: sense, 5′-GAC AAT ATC ATA TAC ATG GAA TGT G-3′;antisense, 5′-GCG GTA GTA ACA GAG AAT TTC TT-3′; and probes 5′-CGA AAATCC AGA ATC GGA ACT TCT T-fluorescein-3′ and 5′-Red 640-CCA TTA CAG TAAACT TTA GGC GGT C-phosphate-3′. Amplification of VZV using such primersdirected toward gene 28 generated a 282 bp amplification product(Saverbrei et al., 1999, J. Clin. Virol., 14:31-6). A PCR master mix(see Espy et al., 2000, J. Clin. Microbiol., 38:795-9) was modified forthe VZV gene 28 LightCycler™ Assay by eliminating DMSO and using 4 mMMgCl and 1 μM gene 28 primers. Samples underwent 45 cycles of:denaturation at about 95° C. immediately followed by primer annealing tothe template nucleic acid for about 12 secs at about 55° C., andelongation of the newly-synthesized strands at about 72° C. for about 12secs.

Primers and probes for detection of VZV DNA using gene 29 were designedusing the OLIGO software (Molecular Biology Insights, Inc., Cascade,Colo.) and had the following sequences: sense, 5′-TGT CCT AGA GGA GGTTTT ATC TG-3′; antisense, 5′-CAT CGT CTG TAA GAC TTA ACC AG-3′; andprobes 5′-GGG AAA TCG AGA AAC CAC CCT ATC CGA C-fluorescein-3′ and5′-Red 640-AA GTT CGC GGT ATA ATT GTC AGT GGC G-phosphate-3′.Amplification using such gene 29 primers produced an amplificationproduct of 202 bp. The PCR master mix (see Espy et al., 2000, J. Clin.Microbiol., 38:795-9) was modified for the VZV gene 29 LightCycler™Assay by using 4 mM MgCl, 1 μM gene 29 primers and 3% dimethylsulfoxide.The thermocycling program for gene 29 was the same as described abovefor gene 28.

Both sets of hybridization probes (i.e., gene 28 and gene 29 probes)contained a donor fluorophore (fluorescein) on the 3′-end of one probe,which when excited by an external light source, emitted light that wasabsorbed by a corresponding acceptor fluorophore (LC-Red 640) at the5′-end of the second hybridization probe. Both the gene 28 and gene 29LightCycler™ assays detected ≧20 genomic copies of VZV.

Example 3 Detection of VZV

Of 253 dermal specimens, VZV was detected in 23 (9.1%) by shell vialcell cultures, while 44 (17.4%) (gene 28) and 50 (19.7%) (gene 29) weredetected by LightCycler™ PCR tests (Table 1). Twenty-one of 44 (47.7%)(gene 28) and 27 of 50 (54.0%) (gene 29) specimens were exclusivelypositive by LightCycler™ PCR; the shell vial cell culture assay wasnever positive when DNA amplification was negative (specificity, 100%).VZV DNA was detected in 39 of 44 (88.6%)(gene 28) and 39 of 50 (78.0%)(gene 29) total specimens positive during cycles 10 through 30 by theLightCycler™ assay. In addition, of the 23 total specimens positive bythe shell vial assay, VZV DNA was detected by both gene 28 and gene 29LightCycler™ assays in these samples by cycle 26, indicating a directrelationship between the capability of culturing the virus by the shellvial assay and the recognition of amplified VZV product in the earlycycles of LightCycler™ PCR.

TABLE 1 Detection of VZV DNA by LightCycler ™ PCR and by shell vial cellculture Number of specimens positive LightCycler ™ Cycle number gene 28gene 29 Shell vial cell culture 0-30 39 39 23 31 0 1 0 32 0 0 0 33 1 2 034 3 4 0 35 1 3 0 36 0 1 0 Total 44 50 23

Of the 50 specimens examined using LightCycler™ PCR and primers andprobes directed toward gene 29, FRET was detected in 11 samples betweencycles 30 and 36. VZV DNA was never exclusively detected using onlyprimers and probes directed toward gene 28 in the absence of a positiveLightCycler™ result using primers and probes directed toward gene 29.Specificity of the LightCycler™ assay was further demonstrated bymelting point analysis, which was performed with all samples from whicha fluorescent signal was generated. All positive samples had a meltingcurve consistent with the positive VZV control.

Primers and probes directed to gene 29 detected 50 specimens containingVZV DNA, whereas 44 specimens were positive for VZV using primers andprobes directed toward gene 28. In routine laboratory practice, primersand probes directed to gene 28 may be used, even though amplificationand detection of VZV using primers and probes directed toward gene 29was more sensitive than that using primers and probes directed towardgene 28. Forty-four samples containing VZV were positive by PCR directedto both gene 28 and gene 29. For clinical implementation, detectionusing PCR amplification directed to either gene 28 or gene 29 is muchimproved over the current assays, although detection of VZV usingprimers and probes directed toward gene 29 is more sensitive. Thus, gene28 or gene 29 can be amplified and detected individually or incombination to detect VZV.

The assay described herein exhibited specificity for VZV sinceVZV-specific primers and probes amplified only VZV DNA. DNA from herpessimplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV),and human herpes viruses 6, 7, and 8 were tested with VZV-specificprimers and probes and were uniformly negative. Significantly, there wasno cross reaction in fluorescence signal between VZV- and HSV-positivesamples.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An article of manufacture, comprising: a pair of VZV gene 29 primers,wherein said pair of gene 29 primers comprises a first gene 29 primerand a second gene 29 primer, wherein said first gene 29 primer consistsof the sequence 5′-TGT CCT AGA GGA GGT TTT ATC TG-3′ (SEQ ID NO:5) andwherein said second gene 29 primer consists of the sequence 5′-CAT CGTCTG TAA GAC TTA ACC AG-3′ (SEQ ID NO:6); a pair of VZV gene 29 probes,wherein said pair of gene 29 probes comprises a first gene 29 probe anda second gene 29 probe, wherein said first gene 29 probe consists of thesequence 5′-GGG AAA TCG AGA AAC CAC CCTATC CGAC-3′ (SEQ ID NO:7) andwherein said second gene 29 probe consists of the sequence 5′-AAG TTCGCG GTA TAA TTG TCA GTG GCG-3′ (SEQ ID NO:8); and a donor fluorescentmoiety and a corresponding acceptor fluorescent moiety.
 2. The articleof manufacture of claim 1, wherein said first gene 29 probe is labeledwith a donor fluorescent moiety and wherein said second gene 29 probe islabeled with an acceptor fluorescent moiety.
 3. The article ofmanufacture of claim 1, further comprising a package insert havinginstructions thereon for using said pair of gene 29 primers and saidpair of gene 29 probes to detect the presence or absence of VZV in abiological sample.